Protein degradation is an essential component in plant growth, development, and environmental responses. For example, under nutrient stress condition, such as nitrogen starvation, old proteins are degraded to supply amino acids for new protein synthesis. Protein degradation is also a hallmark of senescence or apoptosis. Cysteine proteinases (CysP) are involved in various protein degradation pathways.
The invention is based on the discovery of a new promoter derived from a rice CysP gene (designated OsEP3A). The sequence of OsEP3A is shown in FIG. 1. The complete nucleotide sequence of OsEP3A is designated SEQ ID NO:3, while the complete amino acid sequence of the protein encoded by OsEP3A is encoded by SEQ ID NO:4. This sequence begins and ends with an EcoRI site used for cloning as described in the Example below. The upper case nucleotides represent the cDNA sequence, while the lower case nucleotides represent the genomic sequences flanking the cDNA. Nucleotide numberings are relative to the transcription start site. The putative TATA box and polyA signals are underlined. Two putative gibberellic acid (GA3) response elements (GARE) are double underlined. A minimal promoter sequence from xe2x88x92180 to xe2x88x921 is designated SEQ ID NO:1 (bolded sequence), and the promoter-containing genomic sequence upstream of the transcriptional start site (from xe2x88x92851 to xe2x88x921) is designated SEQ ID NO:2. A functional signal peptide (in italics) was identified and designated SEQ ID NO:5.
This new promoter was shown to direct expression of a heterologous protein in the aleurone layer of transgenic rice seeds during germination and in cultured rice suspension cells under nitrogen starvation, but not in the presence of nitrogen, a pattern of expression consistent with the metabolic role of the original CysP gene from which the promoter was derived. Consequently, the promoter can be used to regulate and direct expression of a heterologous protein or RNA in transgenic plants or plant cells.
Accordingly, the invention features an isolated nucleic acid including SEQ ID NO:1 (e.g., SEQ ID NO:2), a promoter that hybridizes under stringent conditions to SEQ ID NO:1, or a promoter that is at least 50% (e.g., at least 60, 70, 80, 90, or 95%) identical to SEQ ID NO:1. The nucleic acid of the invention can further included a heterologous sequence to which a promoter containing SEQ ID NO:1 is operably linked, i.e., the promoter directs transcription of the heterologous sequence. The heterologous sequence can encode a protein or polypeptide, e.g., one having the OsEP3A signal peptide (SEQ ID NO:5). The invention also includes vectors and transformed cells harboring a nucleic acid of the invention, as well as transgenic plants (e.g., a transgenic embryo or germinating seed) whose genomic DNA contains a nucleic acid of the invention. The transgenic plant can be a monocot or dicot. The invention further features a method of producing a transgenic plant or organ of a plant (e.g., a seed) by stably introducing a nucleic acid of the invention into a plant cell, and culturing the plant cell under conditions sufficient for the plant cell to form a plant or organ of a plant.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid which has a non-naturally occurring sequence, or which has the sequence of part or all of a naturally occurring gene but is free of the genes that flank the naturally occurring gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are mixtures of DNA molecules, vectors, or clones as they occur in a DNA library such as a cDNA or genomic DNA library. Also excluded are RNA molecules that consist of naturally-occurring sequences (e.g., naturally-occurring mRNA), except where the RNA is in a purified state such that it is at least 90% free of other naturally-occurring RNA species. Thus, a naturally-occurring mRNA in a whole mRNA preparation prepared from a cell would not be an xe2x80x9cisolated nucleic acid,xe2x80x9d but a single mRNA species purified to 90% homogeneity from that whole mRNA preparation would be.
As used herein, xe2x80x9cpercent identityxe2x80x9d of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
By xe2x80x9chybridizes under stringent conditionsxe2x80x9d is meant specific and non-covalent equilibrium binding by base-pairing to an immobilized reference nucleic acid in a hybridization solution containing 0.2xc3x97SSC (1.75 g/l NaCl, 0.88 g/l Na3citrate.2H2O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68xc2x0 C. Washings, if any are required to achieve equilibrium, are carried out with the hybridization solution.
A xe2x80x9cheterologous sequencexe2x80x9d is a nucleotide sequence that is not naturally operably linked to the OsEP3A promoter in a naturally occurring organism.
A xe2x80x9cpromoterxe2x80x9d is a nucleotide sequence that is capable of directing transcription in at least one context, e.g., when it is operably linked to a heterologous sequence in a plasmid within a plant cell. In other words, a promoter can exist without downstream sequences to transcribe, so long as the promoter sequence can direct transcription when placed upstream of a heterologous sequence in a different context.
The promoter sequence of the invention can be introduced into a variety of plant expression vectors for expressing exogenous proteins in plant cells, transgenic plants, and the aleurone layer of seeds. Such exogenous proteins include anti-ageing or anti-senescent proteins that can prevent tissue damage during stressful conditions, such as during nitrogen starvation. In addition, the isolated nucleic acids of the invention can be used as probes to isolate other promoters and/or genes whose expression is induced under stress or during senescence. For example, nucleotides 18 to 45 (SEQ ID NO:10) or 131 to 170 (SEQ ID NO:11) of SEQ ID NO:1 can be used to screen genomic DNA libraries for genes that are regulated similarly to OsEP3A. Further, the methods of the invention can be used to produce transgenic plants or organs having specialized properties (e.g., longer shelf-life) as a consequence of expressing a heterologous RNA (e.g., a mRNA encoding an anti-ageing protein or an anti-sense RNA that inhibits expression of a senescence-associated gene) in a tissue or organ of a plant.